Hendrychová Tereza, Anzenbacherová Eva, Hudeček Jiří, Skopalík Josef, Lange Reinhard, Hildebrandt Peter, Otyepka Michal, Anzenbacher Pavel
Department of Physical Chemistry, Faculty of Science, Palacky University, Olomouc, Czech Republic.
Biochim Biophys Acta. 2011 Jan;1814(1):58-68. doi: 10.1016/j.bbapap.2010.07.017. Epub 2010 Jul 23.
To gain more complete insight into flexibility and malleability of five forms of human liver cytochrome P450 enzymes, which play major roles in drug metabolism (CYPs 1A2, 2A6, 2C9, 2D6 and 3A4), we employed UV/VIS and resonance Raman spectroscopy in combination with all-atomic molecular dynamics simulations under normal and high pressure conditions (300 MPa). In general, the high pressure reduces the flexibility of CYPs, which become more dense and compact as their radii of gyration and temperature B-factors diminish. The flexibility of CYPs spans the regions, which are localized in solvent exposed loops. A considerable degree of flexibility is also observed at amino-acids making the pw2 and solvent channels, which are suggested to serve for substrate access and/or product release. The number of water molecules as well as the number of protein backbone atoms of the active site in close proximity of heme cofactor generally increases under high pressure. This finding provides new insights regarding the interpretation of pressure-related Soret band red shifts. Presented results also point towards considerable differences between the CYP forms studied: CYP2A6 and CYP1A2 have the least malleable active sites while those of CYP2D6, CYP2C9 and CYP3A4 have considerably greater degrees of flexibility or malleability. In addition, the number of water molecules in the active site cavity of CYP3A4 anomalously decreases under high pressure due to opening of the active site. These results correlate with the known substrate promiscuity of the respective CYP forms, with CYP3A4 displaying the highest substrate promiscuity, corresponding to the most open and malleable active site, whereas CYP1A2 and CYP2A6 show a high substrate-specificity and have a small and rigid active sites.
为了更全面地了解在药物代谢中起主要作用的五种人类肝细胞色素P450酶（CYP 1A2、2A6、2C9、2D6和3A4）的柔韧性和可塑性，我们在常压和高压条件（300 MPa）下，将紫外/可见光谱和共振拉曼光谱与全原子分子动力学模拟相结合进行了研究。一般来说，高压会降低细胞色素P450酶的柔韧性，随着它们的回转半径和温度B因子减小，其结构变得更加致密和紧凑。细胞色素P450酶的柔韧性分布在暴露于溶剂的环区。在构成pw2和溶剂通道的氨基酸处也观察到了相当程度的柔韧性，这些通道被认为用于底物进入和/或产物释放。在高压下，靠近血红素辅因子的活性位点的水分子数量以及蛋白质主链原子数量通常会增加。这一发现为解释与压力相关的Soret带红移提供了新的见解。所呈现的结果还表明所研究的细胞色素P450酶形式之间存在显著差异：CYP2A6和CYP1A2的活性位点可塑性最小，而CYP2D6、CYP2C9和CYP3A4的活性位点具有更高的柔韧性或可塑性。此外，由于活性位点的开放，CYP3A4活性位点腔内的水分子数量在高压下异常减少。这些结果与相应细胞色素P450酶形式已知的底物选择性相关，CYP3A4表现出最高的底物选择性，对应于最开放和可塑性最强的活性位点，而CYP1A2和CYP2A6表现出高底物特异性，且具有小而刚性的活性位点。
